Peptide inhibitors of phospholipase A2 purified from inflammatory sites

ABSTRACT

PCT No. PCT/JP91/01424 Sec. 371 Date Jun. 18, 1992 Sec. 102(e) Date Jun. 18, 1992 PCT Filed Oct. 17, 1991 PCT Pub. No. WO92/06997 PCT Pub. Date Apr. 30, 1992.Peptide inhibitors of phospholipase A2 from inflammatory sites having the amino acid sequence given in formula SEQ. I.D. NO: 1   &lt;IMAGE&gt;  [I]

TECHNICAL FIELD

The present invention relates to peptide inhibitors of phospholipase A₂purified from inflammatory sites.

BACKGROUND ART

Phospholipase A₂ is an enzyme which hydrolyzes β-ester bonds inphospholipid to give fatty acids and lysophospho-lipids. Especially, itreleases arachidonic acid which can be a precursor of prostaglandins,leukotriene, thromboxane and the like from membrane phospholipids and isthought to play an important role in the production of theseinflammatory mediators. In recent years, phospholipase A₂ has beenpurified from the inflammatory sites of human inflammatory diseases andinflammatory model animals (it will be called phospholipase A₂ frominflammatory sites) and its properties have been clarified. This enzymeis thought to have the action to accelerate the inflammatory reactions,therefore the drug to inhibit the activity of this enzyme can beexpected to reveal anti-inflammatory actions.

Complement C3 is a protein which has been known to perform the keyfunctions in the complement pathway. C3 is hydrolyzed stepwise by aprotease in blood. In other words, it is cleaved first with convertaseinto C3a an dC3b. C3b binds through its thiol ester site to the surfaceof activators followed by activation of the complement pathway to form amembrane attack complex. C3a works as an anaphylatoxin. At this time, aminor part of C3b binds to the activators, while the major part reactswith water to lose the binding activity, further undergoes hydrolysis bya protease to convert into C3dg or C3d finally.

The present inventors have already applied for patents after findingthat human and rat C3dg inhibit specifically phospholipase A₂ purifiedfrom inflammation sites, succeeding in expression of rat C3 cDNA inEscherichia coli to produce a part of rat C3α chain (containing the C3dgpart) as a recombinant protein, and realizing that the recombinantprotein inhibits specifically phospholipase A₂ purified frominflammatory sites (PCT/J90/00996, W091/01999).

Human C3dg is, however, a protein of about 39 kDa molecular weight andit can be anticipated that the use of said protein as ananti-inflammatory as such will cause troubles in, for example, thetransference to the affected part, the storage stability orantigenicity.

Thus, a novel peptide having inhibitory activity against phospholipaseA₂ from inflammatory sites is desirably provided as an anti-inflammatorywithout such troubles.

Hereupon, the present inventors have made intensified studies in orderto solve the above-mentioned problems to find that a part of the aminoacid sequence of C3dg has action to inhibit phospholipase A₂, andattained the present invention.

DISCLOSURE OF THE INVENTION

In other words, the present invention is a peptide inhibitor ofphospholipase A₂ purified from inflammatory sites having an amino acidsequence shown in SEQ ID No: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 gives the fractionation of the peptide in Example 1 according tothe present invention by means of reversed phase HPLC.

FIG. 2 gives the inhibitory activity of the peptide according to thepresent invention against phospholipase A₂, which was determined inExample 2.

In these figures, -- show the cases where the phospholipase A₂ purifiedfrom human inflammatory sites, ◯--◯give the phospholipase A₂ from ratinflammatory sites and x--x show the phospholipase A₂ from procinepancreas.

BEST EMBODIMENT OF THE INVENTION

The peptides include the amino acid sequence of 21 residues shown in theSEQ ID No: 1. The amino acid sequence of said peptide is identical withthe amino acid sequence from No. 612 to the C-terminus of human C3αchain. In the amino acid sequence in sequence No. 1, the peptides havingsubstitution, deletion or insertion of one or more amino acid residuesare also included in the peptides according to the present invention aslong as they have the inhibitory activity against phospholipase A₂ frominflammatory sites.

Such peptides according to the present invention can be obtained bysynthesis according to a customary procedure described in, for example,"The basis and experiments in peptide syntheses" (written in Japanese);N. Izumiya, T. Kato, H. Aoyagi and M. Yaki: Maruzen, Tokyo) or E.Atherton, R.C. Sheppard; "Solid phase peptide synthesis, a practicalapproach" (LRL press) followed by purification. Or human C3α chain orthe like is used as a starting substance to be hydrolyzed with an enzymesuch as α-chymotrypsin.

The peptide synthesis, the measurement of the inhibitory activityagainst phospholipase A₂ or the like used in the present invention willbe illustrated in the following:

1Peptide Synthesis and Purification

A peptide synthesizer 431A of Applied Biosystem was employed to conductthe synthesis by the Fmoc method.

The deprotection of the samples was carried out by the TMSBr cleavagemethod according to the protocol of Applied Biosystem.

The samples were purified by using the reversed phase HPLC column (VydacProtein C₁₈, 2.2 cm ID×25 cm L) and eluted with the gradient of 0 to 80%acetontrile in the presence of 0.1% trifluoroacetic acid.

2Determination of Inhibitory Activity Against Phospholipase A₂

The activity-measuring system was prepared by adding distilled water to100 mM Tris-HCl (ph 9.0), 4 mM calcium chloride, 0.1 mMphosphatidylethanolamine (2,000 dpm/nmol), and 10 μl sample to adjustthe total volume to 240 μl, and finally 10 μl of 0.1 ng/μ1 phospholipaseA₂ was added. The phospholipases A₂ used were from human inflammatorysites (the joint fluid from patients with rheumatoid arthritis) (Hara etal., J. Biochem., 104, 326-328, 1988), rat inflammatory sites (Chang etal., J. Biochem., 102, 147-154, 1987), and porcine pancreas (BoehringerManheim Co.). Phosphatidylethanolamine was purified from Escherichiacoli cultured in a medium to which acetic acid was added.

The reactions were conducted at 37° C. with stirring for 10 minutes. Thetermination of the reaction and the extraction of fatty acids generatedwere carried out by the Dole's method (Dole et al., J. Biol. Chem., 235,2595-2599, 1960) and the extracted fatty acid was determined with ascintillation counter.

The present invention will be illustrated in more detail by examples.

EXAMPLE 1 Synthesis and Purification of the Peptides Having InhibitoryActivity Against Phospholipase A₂ from Inflammatory Sites

A peptide was synthesized in accordance with the amino acid sequencegiven in SEQ. ID No: 1 using a peptide synthesizer (Applied Biosystem,413 A). The yield was 77.9%.

After deprotection, the 140.7 mg crude product was fractionated withreversed phase HPLC (FIG. 1). the main peak eluted with about 35%acetonitrile was collected and lyophilyzed. The yield of the finallypurified sample was 21.8 mg. The sample was confirmed to have the aminoacid sequence of No. 1 by a gas-phase protein sequencer 477 A of AppliedBiosystem.

EXAMPLE 2 Phospholipase A₂ Inhibitory Acitivity

The lyophilyzed sample obtained in Example 1 was dissolved again in 50%acetone-0.1% trifluoroacetic acid to adjust the concentration to 10mg/ml. The solution was further diluted stepwise with the same bufferand the inhibitory activity of phospholipase A₂ from inflammatory siteswas determined at individual concentrations.

The results are given in FIG. 2. The peptide according to the presentinvention inhibited phospholipase A₂ from human inflammatory sites(◯--◯) and phospholipase A₂ from rat inflammatory sites (◯--◯)does-dependently. In both enzymes, the amount of the protein needed for50% inhibition of 1 ng enzyme activity was about 300 ng and IC₅₀ wasabout 5×10⁻⁷ M. Meanwhile, it showed no inhibitory activity againstphospholipase A₂ from porcine pancreas (x--x).

FIELD OF INDUSTRIAL UTILITY

Peptide inhibitors of phospholipase A₂ from inflammatory sites accordingto the present invention have inhibitory activity against phospholipaseA₂ from the inflammatory sites and are expected to have an action toinhibit allergic reactions, and can be utilized as an anti-inflammatoryand a therapeutic agent for allergic diseases in mammalians especiallyin human. Additionally, they are expected to have excellent in vivotransference to the affected parts and high storage stability.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnLysAspAlaProAspHisGlnGluLeuAsnLeuAspValSerLeu                              151015                                                                        GlnLeuProSerArg                                                               20                                                                        

We claim:
 1. A purified peptide inhibitor of phospholipase A₂,consisting essentially of the amino acid sequence represented in SEQ IDNo:1.